1. Field of the Invention
This invention relates to immunology, and, more specifically, to the preparation and use of immunopotentiating agents which are capable of eliciting, enhancing and/or otherwise modifying immune responses. These agents, through their ability to elicit or enhance cellular or humoral responses, have potential utility in a variety of disease conditions wherein immunotherapy might be expected to provide a benefit.
2. Description of Related Art
The body's immune system serves as a defense against a variety of conditions, including, e.g., injury, infection and neoplasia, and is mediated by two separate but interrelated systems, the cellular and humoral immune systems. Generally speaking, the humoral system is mediated by soluble products, termed antibodies or immunoglobulins, which have the ability to combine with and neutralize products recognized by the system as being foreign to the body. In contrast, the cellular immune system involves the mobilization of certain cells, termed T-cells, that serve a variety of therapeutic roles.
(a) The Immune System
The immune system of both humans and animals include two principal classes of lymphocytes: the thymus derived cells (T cells), and the bone marrow derived cells (B cells). Mature T cells emerge from the thymus and circulate between the tissues, lymphatics, and the bloodstream. T cells exhibit immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection). T cells act against or in response to a variety of foreign structures (antigens). In many instances these foreign antigens are expressed on host cells as a result of infection. However, foreign antigens can also come from the host having been altered by neoplasia or infection. Although T cells do not themselves secrete antibodies, they are usually required for antibody secretion by the second class of lymphocytes, B cells.
There are various subsets of T cells, which are generally defined by antigenic determinants found on their cell surfaces, as well as functional activity and foreign antigen recognition. Some subsets of T cells, such as CD8.sup.+ cells, are killer/suppressor cells that play a regulating function in the immune system, while others, such as CD4.sup.+ cells, serve to promote inflammatory and humoral responses. (CD refers to cell differentiation cluster; the accompanying numbers are provided in accordance with terminology set forth by the International Workshops on Leukocyte Differentiation (5). A general reference for all aspects of the immune system may be found in (1)).
(b) T Cell Activation
Human peripheral T lymphocytes can be stimulated to undergo mitosis by a variety of agents including foreign antigens, monoclonal antibodies and lectins such as phytohemagglutinin and concanavalin A. Although activation presumably occurs by binding of the mitogens to specific sites on cell membranes, the nature of these receptors, and their mechanism of activation, is not completely elucidated. Induction of proliferation is only one indication of T cell activation. Other indications of activation, defined as alterations in the basal or resting state of the cell, include increased lymphokine production and cytotoxic cell activity.
T cell activation is an unexpectedly complex phenomenon that depends on the participation of a variety of cell surface molecules expressed on the responding T cell population (2,3). For example, the antigen-specific T cell receptor (TcR) is composed of a disulfide-linked heterodimer, containing two clonally distributed, integral membrane glycoprotein chains, .alpha. and .beta., or .gamma. and .delta., non-covalently associated with a complex of low molecular weight invariant proteins, commonly designated as CD3 (the older terminology is T3) (2,4).
The TcR .alpha. and .beta. chains determine antigen specificities (6). The CD3 structures are thought to represent accessory molecules that may be the transducing elements of activation signals initiated upon binding of the TcR .alpha..beta. to its ligand. There are both constant regions of the glycoprotein chains of TcR, and variable regions (polymorphisms). Polymorphic TcR variable regions define subsets of T cells, with distinct specificities. Unlike antibodies which recognize soluble whole foreign proteins as antigen, the TcR complex interacts with small peptidic antigen presented in the context of major histocompatibility complex (MHC) proteins. The MHC proteins represent another highly polymorphic set of molecules randomly dispersed throughout the species. Thus, activation usually requires the tripartite interaction of the TcR and foreign peptidic antigen bound to the major MHC proteins.
With regard to foreign antigen recognition by T cells the number of peptides that are present in sufficient quantities to bind both the polymorphic MHC and be recognized by a given T cell receptor, thus inducing immune response as a practical mechanism, is small. One of the major problems in clinical immunology is that the polymorphic antigens of the MHC impose severe restrictions on triggering an immune response. Another problem is that doses of an invading antigen may be too low to trigger an immune response. By the time the antigenic level rises, it may be too late for the immune system to save the organism.
The tremendous heterogeneity of the MHC proteins among individuals remains the most serious limiting factor in the clinical application of allograft transplantation. The ability to find two individuals whose MHC is identical is extremely rare. Thus, T cells from transplant recipients invariably recognize the donor organ as foreign. Attempts to suppress the alloreactivity by drugs or irradiation has resulted in severe side effects that limit their usefulness. Therefore, more recent experimental and clinical studies have involved the use of antibody therapy to alter immune function in vivo. The first successful attempt to develop a more selective immunosuppressive therapy in man was the use of polyclonal heterologous anti-lymphocyte antisera (ATG) (7, 8, 9).
Clinical trials of the ATG treatment suggested a significant reduction of early rejection episodes, improved long term survival and, most importantly, reversal of ongoing rejection episodes. However, the results were often inconsistent due to the inability to standardize individual preparations of antisera. In addition, the precise nature of the target antigens recognized by the polyclonal reagents could not be defined, thus making scientific analysis difficult. The advent of monoclonal antibody (mAb) technology provided the basis for developing potentially therapeutic reagents that react with specific cell surface antigens which are involved in T cell activation.
(c) Effect of Monoclonal Antibodies on the Immune System
Monoclonal antibodies (mAb) were developed by Kohler and Milstein in 1975. The methods generally used to produce mAb consist of fusing (hybridizing) two types of somatic cells: (1) a neoplastic myeloma cell line; and (2) a normal B lymphocyte obtained from an immunized animal. The result is called a hybridoma which is characterized by immortal growth and the ability to secrete antibodies specific for the immunization antigen.
One of the clinically successful uses of monoclonal antibodies is to suppress the immune system, thus enhancing the efficacy of organ or tissue transplantation. U.S. Pat. No. 4,658,019, describes a novel hybridoma (designated OKT3) which is capable of producing a monoclonal antibody against an antigen found on essentially all normal human peripheral T cells "ATCC Accession No. CRL-8001". This antibody is said to be monospecific for a single determinant on these T cells, and does not react with other normal peripheral blood lymphoid cells. The OKT3 mAb described in this patent is currently employed to prevent renal transplant rejection (10).
One unexpected side effect of the OKT3 therapy was the profound mitogenic effect of the mAb in vivo (28). Although anti-CD3 mAb has been shown to activate T cells in vitro to produce various lymphokines, etc. (11), OKT3 has not been previously used to stimulate the immune system in vivo.
In addition, other cell surface molecules have been identified that can activate T cell function, but are not necessarily part of the T cell surface receptor complex. Monoclonal antibodies against Thy-1, TAP, Ly-6, CD2, or CD28 molecules can activate T cells in the absence of foreign antigen in vitro (12, 13, 14, 15, 16). Moreover, certain bacterial proteins although differing in structure from mabs, also have been shown to bind to subsets of T cells and activate them in vitro (17). Although some of these agents, in vitro effects have previously been demonstrated, in vitro activity is often not a reliable predictor of in vivo effects.
(d) Immune System and Tumor Growth
One cause of malignant tumor growth is believed to be the inability of the immune system to respond appropriately to tumor antigen. For example, malignant progressor tumors are only weakly immunogenic and can evade host recognition and rejection. Both specific and non-specific effector pathways have been implicated in tumor immunity. Treatment by immunotherapy is aimed at remedying defects in the immune weaponry. The aim of immunotherapy has been the enhancement of one or both of these pathways. One potential approach to therapy is to activate host antitumor cellular effector mechanisms.
Historically, non-specific adjuvants such as BCG or pertussis have been used to augment immune responses. In normal individuals these adjuvants amplify immune responses by providing non-specific stimuli that enhance overall immunity. However, these adjuvants do not selectively act on T cells, or subsets of T cells, and have not been shown capable of overcoming immunodeficiency states. Unfortunately, current modes of immunotherapy which induce non-specific effector cells are not effective enough in potentiating anti-tumor responses (18). Recently, immunotherapy regimens which utilize the ability of the immune system to recognize tumor antigens in a specific manner, for instance utilizing specific tumor-infiltrating lymphocytes, for immunotherapy, have been suggested to result in superior anti-tumor immunity (19). Thus, current efforts toward developing more efficacious forms of immunotherapy have focussed on specific anti-tumor response and memory-following antigen recognition. One approach that has not previously been accomplished has been the in vivo administration of T cell activating mAbs to promote anti-tumor activity.
(e) Defenses Against Viral Infections
Human immunodeficiency virus (HIV), the biologic agent of AIDS, causes a persistent infection associated with profound immunosuppression resulting in susceptibility to opportunistic infections. Immunological responses to HIV infection require the development of both humoral and cell mediated effector mechanisms; however current efforts in treatment and vaccine design have fallen short of success either due to the immunodeficiency associated with the viral infection, or to the low immunogenicity of the vaccine (20). The development of a safe and effective vaccine against infection with human immunodeficiency virus (HIV) is complicated by a lack of understanding of protective immunity to HIV and disease development, and the absence of an adequate and convenient animal model for studying HIV infection.
Because HIV can be transmitted as either a cell-free or cell-associated virus, a protective immune response against HIV infection will likely require both humoral and cell-mediated immunity, including neutralizing antibody against HIV, antibodies involved in antibody-dependent cellular cytotoxicity and cytotoxic lymphocytes. All of these activities involve virus-specific T cells. T cell activation requires potent in vivo immune responses to foreign antigens such as viruses.
In individuals infected with HIV, two components of the immune system are suboptimal and, therefore, the ability to generate an immune response in these individuals has been compromised. First, the reduced frequency of antigen-reactive CD4.sup.+ T cells is apparently not sufficient to mount an appropriate immune response to HIV, especially if the quantity of HIV antigen is low. CD4 is a membrane protein that acts as a binding site and entry port into CD4.sup.+ lymphocytes for the human immunodeficiency virus-type 1 (HIV-1) (21). Second, all immune responses are dependent on the ability of T cells to recognize processed antigen associated with major histocompatibility antigens (MHC). Any vaccine approach which utilizes HIV peptides or inactivated virus antigen must depend on the ability of antigenic peptides to bind the appropriate MHC antigens necessary to initiate an immune response. Given the tremendous polymorphism of the MHC antigens expressed in the population, and the variation of the HIV virus, developing a successful HIV vaccine for general use is difficult and has not yet been successful.
(f) Problems in Developing Vaccines to Weakly Immunogenic Antigens
The usefulness of certain peptides, proteins or other potential or desired immunogens in vaccines can be limited by several critical factors. For example, low immunogenicity of the peptide or other structure which one desires to employ can be a difficult problem to overcome, particularly with smaller peptides and those peptides which do not contain appropriately strong B- and/or T-cell potentiating sequences. Such peptides are typically only weakly immunogenic at best. Moreover, to be of widespread applicability, the peptides chosen must be capable of inducing an immune response in a majority of the population.
It has been difficult to protect against attack by organisms such as the HIV virus or to provide tumor immunity for several reasons. For example, genetic differences exist among individuals at the major histocompatibility locus, which limits the system's ability to respond to individual small peptides. Thus, the various components of the immune response, including the T cells and B cells, may not interact appropriately in generating a response to non- or weakly-immunogenic small peptides. Attempts to improve peptidyl immunogenicity have centered principally on the use of adjuvants such as alum or complete Freund's adjuvant. However, prior adjuvants such as these have proven to be inadequate for various reasons, including an inability of the adjuvant to specifically enhance T or B cell activity and the inability of the adjuvant to overcome the severe limitations of MHC restriction.
Although glimpses into the defense mechanisms of the body's own immune system have been provided by in vitro studies and by observation of some in vivo reactions, there is a serious lack of successful therapeutic methods to augment immunity in vivo. Improved compositions and/or methodology for eliciting or enhancing cellular or humoral responses in mammals are needed both to provide animal models for investigation of therapeutic regimes, to provide novel means of preparing improved immune system-directed products such as improved immunotherapeutic antibodies, and to advance treatment and possible immunization, e.g., for conditions such as HIV, cancer and infections.